Through an integrated molecular- and histopathology-based screening system, we performed a screening for fusions of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1, receptor tyrosine kinase (ROS1) in 1,529 lung cancers and identified 44 ALK-fusion-positive and 13 ROS1-fusion-positive adenocarcinomas, including for unidentified fusion partners for ROS1. In addition, we discovered previously unidentified kinase fusions that may be promising for molecular-targeted therapy, kinesin family member 5B (KIF5B)-ret proto-oncogene (RET) and coiled-coil domain containing 6 (CCDC6)-RET, in 14 adenocarcinomas. A multivariate analysis of 1,116 adenocarcinomas containing these 71 kinase-fusion-positive adenocarcinomas identified four independent factors that are indicators of poor prognosis: age ≥ 50 years, male sex, high pathological stage and negative kinase-fusion status.

Purpose: EML4-ALK is a transforming fusion tyrosine kinase, several isoforms of which have been identified in lung cancer. Immunohistochemical detection of EML4-ALK has proved difficult, however, likely as a result of low transcriptional activity conferred by the promoter-enhancer region of EML4. The sensitivity of EML4-ALK detection by immunohistochemistry should be increased adequately. Experimental Design: We developed an intercalated antibody-enhanced polymer (iAEP) method that incorporates an intercalating antibody between the primary antibody to ALK and the dextran polymer-based detection reagents. Results: Our iAEP method discriminated between tumors positive or negative for EML4-ALK in a test set of specimens. Four tumors were also found to be positive for ALK in an archive of lung adenocarcinoma (n = 130) and another 4 among fresh cases analyzed in a diagnostic laboratory. These 8 tumors were found to include1with EML4-ALK variant1,1with variant 2, 3 with variant 3, and 2 with previously unidentified variants (designated variants 6 and 7). Inverse reverse transcription-PCR analysis revealed that the remaining tumor harbored a novel fusion in which intron 24 of KIF5B was ligated to intron 19 of ALK. Multiplex reverse transcription-PCR analysis of additional archival tumor specimens identified another case of lung adenocarcinoma positive for KIF5B-ALK. Conclusions: The iAEP method should prove suitable for immunohistochemical screening of tumors positive for ALK or ALK fusion proteins among pathologic archives. Coupling of PCR-based detection to the iAEP method should further facilitate the rapid identification of novel ALK fusion genes such as KIF5B-ALK.Gene fusion is a major mechanism of carcinogenesis in hematologic malignancies and sarcomas (1). Identification of the BCR-ABL fusion kinase, which is generated as a result of the balanced chromosome anomaly t(9;22)(q34;q11) in chronic myelogenous leukemia (2), has thus been followed by the discovery of many fusion-type oncogenes (3). In contrast, it has remained unclear whether such translocationdependent fusion-type oncogenes also play a major role in the pathogenesis of epithelial tumors. Recently, however, almost 50% of prostate cancer cases have been suggested to harbor gene fusions involving ETS transcription factor loci (4), and we have discovered a recurrent chromosome translocation, inv(2)(p21p23), in non -small cell lung cancer (NSCLC) that results in the production of an EML4-ALK fusion-type protein tyrosine kinase (PTK;.Forced expression of EML4-ALK in lung epithelial cells induced the rapid development of hundreds of lung cancer nodules in mice, and peroral administration of inhibitors of the PTK activity of EML4-ALK was shown to clear such tumors from the lungs, demonstrating the pivotal role of EML4-ALK in the pathogenesis of NSCLC positive for this fusion kinase (9). This latter observation also supports the clinical application of ALK

A subset of lung cancers harbors a small inversion within chromosome 2p, giving rise to a transforming fusion gene, EML4-ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene), which encodes an activated tyrosine kinase. We have earlier examined the presence of EML4-ALK by multiplex reverse transcription-polymerase chain reaction in 363 specimens of lung cancer, identifying 11 adenocarcinoma cases featuring the fusion gene. In this study, we clinicopathologically examined the characteristics of the EML4-ALK-positive cases, including the mutation status of EGFR, KRAS, and TP53, and whether they were of thyroid transcription factor-1 (TTF-1) cell lineage or not. Of 11 patients, 4 (36%) with EML4-ALK-positive lung adenocarcinomas who were below 50 years of age were affected by these diseases, as compared with 12 of 242 patients (5.0%) with EML4-ALK-negative lung adenocarcinomas (P ¼ 0.00038). EML4-ALK-positive lung adenocarcinomas were characterized by less-differentiated grade (P ¼ 0.0082) and acinar-predominant structure (Po0.0001) in histology. Furthermore, the presence of EML4-ALK appears to be mutually exclusive for EGFR and KRAS mutations (P ¼ 0.00018), whereas coexisting with TP53 mutations at a low frequency (1/11 ¼ 9.1%), and correlating with non-or light smoking (P ¼ 0.040), in line with the TTF-1 immunoreactivity. Thus, EML4-ALK-positive tumors may form a distinct entity among lung adenocarcinomas, characterized by young onset, acinar histology, no or rare mutations in EGFR, KRAS, and TP53, and a TTF-1 cell lineage, all in agreement with the prevalence in non-or light smokers.

Purpose: EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2)(p21p23) in the genome of non^small cell lung cancer (NSCLC).To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes. Experimental Design: Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs. Results: From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens. Conclusions:These data reinforce the importance of accurate diagnosis of EML4-ALK^positive tumors for the optimization of treatment strategies.

In the present first investigation of EML4-ALK fusion in a large study of lung cancers (5/221), we found an interesting histotype-genotype relationship. Furthermore, we could detect the fusion protein by immunohistochemistry, pointing to possible clinical applications.

Adenocarcinomas with a micropapillary pattern (MPP), featuring small papillary tufts lacking a central fibrovascular core, are thought to have a poor prognosis. To examine whether the MPP is a predictor of prognosis, clinicopathologic characteristics of adenocarcinomas were analyzed with particular reference to survival of early-stage patients. The subjects were 344 consecutive patients (female/male ratio 163:181) for whom complete surgical resection was undertaken at the Cancer Institute Hospital, Japan, during 1986-1995. Histologically, they were divided into two groups: MPP-positive (n = 139; 40%) and MPP-negative (n = 205; 60%). The following items were significantly more frequent in the MPP-positive group: metastasis to lymph nodes (p <0.001), pleural invasion (p = 0.02), intrapulmonary metastasis (p <0.001), and nonsmoking status (p = 0.002). In stage I patients (i.e., without lymph node metastasis, n = 154), 5-year survival of the MPP-positive group (n = 45) was 79%, significantly lower than the MPP-negative group (n = 109) of 93% (p = 0.004). In many cases of the c-stage I MPP-positive group, upstaging was necessary on the basis of pathologic findings for metastases, and the survival was between stage I and stage II. Our study clearly indicated that the MPP is a distinct prognostic marker for lung adenocarcinoma, particularly regarding apparent stage I diseases.

Enhanced glucose and lipid metabolism is one of the most common properties of malignant cells. ATP citrate lyase (ACLY) is a key enzyme of de novo fatty acid synthesis responsible for generating cytosolic acetyl-CoA and oxaloacetate. To evaluate its role in lung cancer progression, we here analyzed ACLY expression in a subset of human lung adenocarcinoma cell lines and showed a relationship with the phosphatidyl-inositol-3 kinase-Akt pathway. The introduction of constitutively active Akt into cells enhanced the phosphorylation of ACLY, whereas dominant-negative Akt caused attenuation. In human lung adenocarcinoma samples, ACLY activity was found to be significantly higher than in normal lung tissue. Immunohistochemical analysis further showed phosphorylated ACLY overexpression in 162 tumors, well-correlating with stage, differentiation grade, and a poorer prognosis. Finally, to show the therapeutic potential and mechanism of ACLY inhibition for lung cancer treatment, we assessed the effect of RNA interference targeting ACLY on lipogenesis and cell proliferation in A549 cells. ACLY inhibition resulted in growth arrest in vitro and in vivo. Interestingly, increased intracellular lipids were found in ACLY knockdown cells, whereas de novo lipogenesis was inhibited. Supplementation of insulin could rescue the proliferative arrest elicited by ACLY inhibition; however, in contrast, fatty acid palmitate induced cell death. Taken together, these findings suggest that ACLY is involved in lung cancer pathogenesis associated with metabolic abnormality and might offer a novel therapeutic target. [Cancer Res 2008;68(20):8547-54]

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